In both children and adults, wound repair proceeds in a cascade of four overlapping major stages: hemostasis, inflammation, proliferation (fibroplasia, angiogenesis, formation of granulation tissue), and scar maturation [5, 6]. In all postnatal animals (except certain amphibians), synthesis and remodeling of the new extracellular matrix in wounds lead to scarring (Table 3.1).

Hemostasis is the first phase of the postnatal or adult wound-healing response. Whether caused by a scalpel or burn, tissue injury leads to vascular disruption and extravasation of red blood cells, platelets, and plasma. Tissue thromboplastin and activated factor XII cause activation of the coagulation cascades and formation of a hemostatic platelet-fibrin plug (Fig. 3.2). Within the wound plug, aggregated platelets become activated after binding to fibronectin, collagen, and other wound proteins [7]. Platelets then degranulate and release proteases, vasoactive amines, and adhesive glycoproteins, such as fibrinogen and fibronectin. In addition, platelets release a cadre of biologically active growth factors, such as platelet-derived growth factor (PDGF), transforming growth factor-beta-1 (TGF-β1), and insulin-like growth factor-1 (IGF-1). Aside from limiting blood loss, the platelet-fibrin clot also forms a protective scab. Furthermore, fibrin and fibronectin within the clot form a provisional matrix that provides a scaffold for monocyte, fibroblast, and keratinocyte deposition [8].

In the inflammatory/migratory phase of adult repair, white blood cells enter the area of injury [5, 9]. Neutrophils are recruited first via chemotactic stimulus to scavenge cellular debris, dirt, and bacteria. Neutrophils are the most predominant cell type during the first 48 h after injury in adults (Fig. 3.2). However, it has been shown that the absence of neutrophils does not prevent healing. In fact, mice studies have shown that neutrophils are not essential to the wound-healing process [10]. The expression of adhesion molecules is upregulated to facilitate passage of leukocytes from the blood stream into the wound [11]. Subsequently, cellular degradation products attract monocytes to the wound, and, along with macrophages, they synthesize a plethora of growth factors [PDGF, TGF-β, fibroblast growth factors (FGF), and transforming growth factor-alpha (TGF-α)]. These growth factors attract and activate local fibroblasts, endothelial cells, and epithelial cells to initiate their respective roles in the wound-repair process. As opposed to neutrophils, tissue macrophages are essential in wound repair. Not only do macrophages remove debris, they also secrete collagenases and other enzymes that break down necrotic tissue [12]. Macrophages provide the signaling cascade that initiates angiogenesis and fibroblasts to regenerate the dermis. Further, macrophages can and do produce many of the matrix elements and matrix-remodeling metalloproteinases [13, 14].

In the proliferative and synthetic phases of wound healing, growth of new tissue occurs. During this phase, granulation tissue formation, angiogenesis, epithelialization, and fibroplasia restore tissue integrity and function. Within hours of skin injury, epidermal cells at the wound margin acquire a flattened phenotype and pseudopodia in preparation for wound contraction and migration across the tissue defect [15]. In addition, migrating keratinocytes upregulate expression of specific cell surface receptors that bind to extracellular molecules within the wound matrix, such as hyaluronic acid, fibronectin, fibrin, and collagens [16–18]. Growth factors regulate reepithelialization [5, 15, 19]. TGF-β and epidermal growth factor (EGF) stimulate keratinocyte migration and synthesis of fibronectin. EGF and TGF-α appear to mediate epidermal cell proliferation at the wound margin [20]. Once epithelial integrity is restored, basement membrane proteins (laminin, collagen) reappear, and keratinocytes reform hemidesmosomes and other cell-cell and cell-matrix attachments.

Angiogenes is is the formation of new blood vessels, or neovascularization. It occurs in concert with reepithelialization and granulation tissue formation [5, 9, 21]. Endothelial cells at the tip of injured capillaries form new capillary buds and migrate into the wound in response to several factors (fibronectin, heparin, PDGF, basic FGF, and vascular endothelial growth factor (VEGF)). Low oxygen tension, lactic acid, and biogenic amines further potentiate angiogenesis. Basic FGF and heparin act in synergy to enhance new vessel growth.

Within 48–72 h after injury, granulation tissue begins to form during the proliferative phase of wound healing. Granulation tissue consists of a tissue matrix hosting a variety of cell types. Fibroblasts from the surrounding tissue migrate into the wound, proliferate, and begin to synthesize extracellular matrix molecules by excreting collagen and fibronectin [5, 21]. Macrophage-derived growth factors , such as TGF-β, stimulate fibroblast proliferation and increased synthesis of collagen, elastin, and proteoglycans. During wound remodeling, fibroblasts secrete hyaluronidase to break down and replace the provisional hyaluronate-rich matrix with larger sulfated glycosaminoglycans. With further wound remodeling, type I collagen gradually replaces type III collagen as the primary collagen in scarring.

Subpopulations of fibroblasts may serve different functions in the wound-repair process. Myofibroblasts are specialized mesenchymal cells that share functional and structural characteristics of both fibroblasts and smooth muscle cells. They express a type of actin called α-smooth muscle actin , a cytoskeletal microfilament that functions in smooth muscle type contraction using the myosin-actin complex [22–24]. Myofibroblasts are naturally found in some subepithelial mucosal surfaces such as the gastrointestinal or genitourinary tract. They provide support and have paracrine function. They were first described in granulation tissue and appear transiently to mediate wound contraction, collagen synthesis, and scar formation. They help speed up wound repair by contracting the edges of the wound. After wound healing is complete, and during the maturation phase, collagen is realigned along tension lines, and myofibroblasts undergo apoptosis; however they may occasionally persist and contribute to the formation of keloids or hypertrophic scars. Myofibroblasts additionally persist in many fibrotic diseases, such as hepatic cirrhosis, renal and pulmonary fibrosis, Dupuytren contracture, and neoplasia-induced desmoplastic reactions. Understanding the signals that regulate myofibroblast apoptosis may contribute to our understanding of hypertrophic scars and how to prevent their formation.

In newborn and adult animals alike, matrix synthesis and remodeling invariably lead to scar formation [5, 9, 21]. Although scar remodeling occurs for months to years after the initial injury, complete restoration of the normal extracellular matrix architecture is never achieved [6]. Mature scars restore only 70% of the tensile strength of normal skin, and normal function is never completely recovered [25]. In general, scarring refers to alteration in the normal composition and organization of a tissue. Scarring is perhaps best characterized in skin, where it refers to a disruption in the pattern of collagen and other extracellular proteins of the dermis. A mature scar is a relatively acellular, avascular area with abundant parallel bundles of collagen, a lack of elastin, and an absence of dermal appendages such as sweat glands and hair follicles. The size of a scar depends on the extent of tissue injury, the degree of wound contamination, the inflammatory response, and the overall nutritional status of the host [9]. A clean surgical incision in which the wound edges are carefully reapproximated heals with minimal scarring. Larger, contaminated wounds result in larger scars.

In contrast, the embryo and early gestation fetus can heal skin wounds without scarring and with regeneration of dermal appendages and restoration of a normal pattern of collagen and extracellular matrix that is indistinguishable from the surrounding, unwounded skin. Although many differences between fetal and adult wound repair have been described, the key mechanisms that mediate scarless fetal repair remain unclear. In the next sections of this chapter, we review those unique characteristics of the fetus that may contribute to scarless repair as well as some new advents in the field.

Several fundamental differences exist between fetal and adult skin that may be at the origin of the scar-free fetal tissue repair. Understanding skin embryogenesis can provide insights into the major differences between fetal and adult scarring. In the human embryo, a primitive epidermis first appears at gestational day 20. During 4–8 weeks of gestation, the human primitive epidermis develops into two layers, the periderm and the basal cell layer. Epidermal stratification begins by 9 weeks, and epidermal keratinization begins by 21 weeks. By 16 weeks, fetal skin has many of the adult skin components such as a basal cell layer, an intermediate layer, sweat glands, hair follicles, and follicular keratinization. By 24 weeks gestation, human fetal skin has all the features of mature adult skin, and at birth neonatal skin is histologically indistinguishable from adult skin [26–28].

Scarring in adult wounds occurs mainly at the dermal level. The fetal dermis similarly undergoes morphologic and biochemical changes during the healing process however without major structural alterations [26]. Early in gestation, the fetal dermis is thin and cellular with a paucity of extracellular matrix. As development proceeds, dermal collagen is deposited, and sulfated glycosaminoglycans replace hyaluronic acid and other non-sulfated glycosaminoglycans. The rapid growth rate of fetal skin and the loose, hyaluronic acid-rich composition of the extracellular matrix provide a conducive environment for scarless fetal repair. Scarless fetal wound healing was first described about three decades ago [29]. It was demonstrated by the repair model of full-thickness eyelid colobomas produced in midgestational fetal sheep (Fig. 3.3). The same occurs in humans before 24 weeks GA [2].

It has been suggested that differences between the fetal and adult wound environments may influence wound repair. Fetal wounds are bathed in warm, sterile amniotic fluid that is rich in growth factors and certain adhesion molecules, such as fibronectin and hyaluronic acid [30]. Furthermore, fetal subcutaneous tissue is relatively hypoxemic as compared to the adult counterpart. However, early evidence suggests that the fetal environment is neither sufficient nor necessary for scarless fetal repair as fetal skin outside the uterine environment also heals without scar as demonstrated by experiments on the newborn opossum [31, 32]. At birth, the opossum remains fetal-like, both physiologically and anatomically, and remains attached to the mother’s nipple for 4–5 weeks. Despite their extrauterine location, incisional wounds in early pouch young reepithelialize within 2 days, synthesize collagen within 24 h, and heal scarlessly [31]. In contrast, wounds in older pouch young heal more slowly and with scarring. Furthermore, human fetal skin transplanted subcutaneously to an immuno-incompetent mouse, such as the athymic nude mouse, retains its developmental characteristics [27] and heals scarlessly [33]. Thus, scarless repair capabilities appear to be intrinsic to the fetal tissue itself, rather than dependent on the sterile, in utero amniotic environment. On the other hand, adult skin in the fetal environment heals with scarring. When adult sheep skin is transplanted into 60-day-gestation fetal sheep (before term which is 145 days and before the fetal immune system has developed sufficiently to reject the allografts), incisional wounds in the adult grafts heal with scarring [34]. Thus, scar formation appears to be intrinsic to adult skin, and neither the amniotic fluid environment nor perfusion by fetal blood prevented scarring. Late gestation fetuses have been observed to heal with scarring despite remaining within the amniotic environment. Primate fetuses demonstrate these developmental changes in their repair response [35]. Full-thickness, upper lip wounds in fetal rhesus monkeys at 75 days GA (165 days being full term) heal scarlessly with regeneration of hair follicles and a dermal collagen pattern that is indistinguishable from unwounded tissue (Fig. 3.4). Conversely, similar wounds in fetuses at 107 days GA or older heal with scarring. Interestingly, wounds made in 85–100 day gestation fetuses heal with intermediate, transitional repair morphology. Fetal wound-healing studies in rodents [36], opossum [32], and sheep [37] demonstrate a similar developmental transition from scarless to scar-type repair. Thus, there is more to the mechanisms of scarless fetal repair than the sterile, growth factor-rich amniotic environment.

Not all fetal organ systems heal scarlessly. Whereas fetal skin, bone, and cartilage can regenerate, fetal nerve tissue, diaphragm, stomach, and peritoneum scar [30, 38–41]. Additionally, scarring is positively correlated with wound size, and while small fetal wounds heal scarlessly, large fetal wounds scar [42]. The unique characteristics of fetal tissue repair appear to be intrinsic to fetal skin and the cells and extracellular matrix that compose it.

Fetal-type wound healing is most remarkable for its regenerative speed and the absence of scarring, even at the histologic level. All primary histologic structures (hair follicles and sweat glands) are restored in the process. Studies reveal that the differences between fetal and adult type of wound repair lie mainly in the inflammatory and proliferative phases [33]. Additionally, differences are noted both at the intracellular level and extracellular level/matrix (Table 3.1).